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Damage induced by stannous chloride in plasmid DNA
Toxicology Letters 116 (2000) 159 – 163 www.elsevier.com/locate/toxlet
Damage induced by stannous chloride in plasmid DNA Jose´ C.P. de Mattos a, Fla´vio J.S. Dantas a, Roberto J.A.C. Bezerra a, Mario Bernardo-Filho a, Janua´rio B. Cabral-Neto b, Claudia Lage b, Alvaro C. Leita˜o b, Adriano Caldeira-de-Arau´jo a,* a
Departamento de Biofı´sica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Uni6ersidade do Estado do Rio de Janeiro, A6. 28 de Setembro, 87, Rio de Janeiro, RJ 20551 -030, Brazil b Laborato´rio de Radiobiologia Molecular, IBCCF, CCS, UFRJ, Rio de Janeiro, RJ 21949 -900, Brazil Received 6 March 2000; received in revised form 2 May 2000; accepted 3 May 2000
Abstract Stannous chloride (SnCl2) is widely used in daily human life, for example, to conserve soft drinks, in food manufacturing and biocidal preparations. In nuclear medicine, stannous chloride is used as a reducing agent of Technetium-99m, a radionuclide used to label different cells and molecules. In spite of this, stannous chloride is able to generate reactive oxygen species (ROS) which can damage DNA. In this work, plasmid DNA (pUC 9.1) was incubated with SnCl2 under different conditions and the results analyzed through DNA migration in agarose gel electrophoresis. Our data reinforce the powerful damaging effect induced by stannous ion and suggest that this salt can play a direct role in inducing DNA lesions. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Stannous chloride; Reactive oxygen species; DNA damage; Plasmid DNA
1. Introduction Reactive oxygen species (ROS) can be generated by several physical and chemical agents. The mono-electronic reduction of ground state oxygen (which occurs during ATP synthesis) causes the appearance of oxygen free radicals, which are implicated in the induction of DNA lesions (Meneghini, 1988; Ferguson, 1994). In view of the importance of DNA damage in carcinogenesis, it is reasonable to suppose that any agent capable of * Corresponding author. Fax: +55-21-2543532. E-mail address: [email protected] (A. Caldeira-de-Arau´jo).
reacting with DNA and modifying its structure could be potentially carcinogenic. Thus, oxidative DNA lesions would account for increasing risks of cancer development. Studies on the genotoxic properties of SnCl2 revealed that it can generate ROS and breaks in DNA (Caldeira-de-Arau´jo et al., 1996) and induces lethality in Escherichia coli, whose damage recovery depends on RecA-mediated repair (Bernardo-Filho et al., 1994). Humans are exposed to stannous chloride (SnCl2) present in packaged food, soft drinks, biocides, dentifrices, etc. (Hallas and Cooney, 1981; McLean et al., 1983; Rader, 1991; White,
0378-4274/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 0 ) 0 0 2 1 3 - 7
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J.C.P. de Mattos et al. / Toxicology Letters 116 (2000) 159–163
1995; Budavery, 1996). In addition, SnCl2 is used in nuclear medicine procedures to obtain Technetium-99m-labelled radiopharmaceuticals (Bernardo-Filho et al., 1994; Caldeira-de-Arau´jo et al., 1996). ROS like hydroxyl radicals (OH) can be produced through the Fenton reaction, i.e. the reduction of superoxide anion (O2−) by Fe2 + ions. Cu2 + and other metal ions are also able to produce O2− and OH through a Fenton-like reaction (Loeb et al., 1988; Simpson et al., 1988; Shi et al., 1994). Dantas et al. (1996) showed that SnCl2-induced lesions could also be generated by a Fenton-like reaction. Moreover, stannous ions have mutagenic effects, which are suggested to occur mainly via DNA oxidation and production of 8-hydroxyguanine (8-oxoG) (Cabral et al., 1998). In addition, Caldeira-de-Arau´jo et al. (1996) showed increased survival of E. coli treated with SnCl2 in the presence of catalase, ROS scavengers or metal ion chelators. In light of these data, we decided to investigate, at a molecular level, using isolated pUC 9.1 plasmid DNA, the possible direct and indirect effects of stannous ions. Conformational changes induced by SnCl2 in plasmid DNA were analyzed in agarose gel electrophoresis. This protocol had already been used to demonstrate the induction of single strand breaks (SSB) by singlet oxygen (Devasagayam et al., 1995). Our data are in accordance with the hypothesis that oxidative lesions can be induced by SnCl2, but also suggest a kind of damage that may be induced by tin itself.
Fig. 1. Agarose gel electrophoresis of plasmid DNA (pUC 9.1, 200 ng) treated with 200 mg/ml SnCl2 for different periods of time. Lanes: (1) control; (2) 30 min; (3) 60 min (4) l Hind III DNA, 250 ng.
2. Material and methods Plasmid DNA (pUC 9.1) was obtained by alkaline lysis as described in Sambrook et al. (1989) and used elsewhere (Caldeira-de-Arau´jo et al., 1996; Assis et al., 1998; Felzenszwalb et al., 1998). Electrophoresis were carried out in 0.8% agarose gels (Sambrook et al., 1989) as previously described (Caldeira-de-Arau´jo et al., 1996; Assis et al., 1998; Felzenszwalb et al., 1998). Plasmid DNA was treated with SnCl2.2H2O (Sigma, St Louis, MO) at 200 mg/ml, for varying periods of time, at room temperature, according to the experimental purpose. Hind III restriction enzyme (Gibco BRL, USA) was used to linearize plasmid DNA when necessary. In some experiments, nitrogen atmosphere was employed to diminish the amount of ROS induced by SnCl2. Ultrapure water (Milli-Q system) was used as solvent. Aliquots of each sample (200 ng DNA) were applied in a horizontal gel plate and electrophoresed at 6 V/cm. The gels were stained with ethidium bromide (0.5 mg/ml) and photographs were taken with a Polaroid camera. Each assay was repeated, at least, three times, the best photos were scanned and the bands quantified through the Gel-Pro Analyzer 3.0 computer program.
3. Results and discussion Several unwanted effects of stannous chloride have been described in the literature (Johnson et al., 1982; Larsson et al., 1990; Hattori and Maehashi, 1994). In the past few years, cytotoxic and genotoxic SnCl2-induced damage were demonstrated in E. coli and the effects appeared to be mediated by reactive oxygen species (Caldeira-deArau´jo et al., 1996; Dantas et al., 1996; Assis et al., 1998; Felzenszwalb et al., 1998; Dantas et al., 1999; Reiniger et al., 1999). Results shown in Fig. 1 indicate that SnCl2 modifies plasmid DNA conformational structure (supercoiled form I changed to open circle form II) in a time-dependent manner. Conversely, if SnCl2 is allowed to undergo pre-oxidation, before it is added to DNA, the number of induced plasmid DNA SSB decreases. This effect was seen
J.C.P. de Mattos et al. / Toxicology Letters 116 (2000) 159–163
Fig. 2. Agarose gel electrophoresis of plasmid DNA (pUC 9.1, 200 ng) treated with 200 mg/ml SnCl2 for 30 min after different periods of exposure of stannous chloride solution to air. Lanes: (1) control; (2) 0 h; (3) 2 h; (4) 4 h; (5) 24 h; (6) l Hind III DNA, 250 ng. Table 1 Quantification of supercoiled form through Gel-Pro Analyzer computer programa Percent of supercoiled form in lane
Fig. 1 Fig. 2 Fig. 4
1
2
3
4
5
100.00 100.00 100.00
49.10 32.10 30.66
41.20 35.20 41.00
– 40.68 100.00
– 75.15 –
a Photos of the gels were scanned and the form I was quantified using a l Hind III DNA, 250 ng as standard. Control samples were normalized as 100% supercoiled form.
Fig. 3. Agarose gel electrophoresis of plasmid DNA (pUC 9.1, 200 ng) linearized with Hind III restriction enzyme and treated with different SnCl2 concentrations for 1 h. Lanes: (1) control; (2) 50 mg/ml SnCl2; (3) 200 mg/ml SnCl2; (4) 400 mg/ml SnCl2; (5) 800 mg/ml SnCl2; (6) 1600 mg/ml SnCl2; (7) control; (8) molecular weight standard DNA (200 ng).
in an agarose gel electrophoresis of SnCl2-treated DNA after exposure of stannous chloride solution to air for different periods of time (Fig. 2). The percentage of supercoiled form I after each experimental situation was quantified by means of a
161
computer program (Gel-Pro Analyzer) and the data are presented in Table 1. These results (Figs. 1 and 2), combined with those from Table 1, can be explained if we assume that stannous chloride has oxidative properties. The ROS generated were responsible for the increase in the number of DNA breaks, in a timedependent manner, as shown in Fig. 1 and confirmed by the decrease in the percentage of supercoiled form (Table 1). The number of DNA SSB decreased when the stannous chloride solution was maintained at room temperature before incubation with plasmid DNA, once more showing the importance of ROS in the induction of DNA lesions (Fig. 2 and Table 1). A stannous chloride pre-oxidation period in periods of time lower than 2 h did not change plasmid DNA electrophoretic profiles (data not shown). An interesting observation that can be seen in Fig. 2 (lane 5) is that even after 24 h of pre-oxidation of the SnCl2 solution, there is a residual amount of DNA lesions in plasmid DNA. It suggests another kind of way that lesions could be produced through a possible direct effect of SnCl2. In order to clarify this point, we carried out experiments in which pUC 9.1 plasmid DNA was first linearized with Hind III restriction enzyme and then treated with increasing concentrations of SnCl2. Fig. 3 shows a crescent delay in DNA migration through agarose gel with increasing amounts of SnCl2. The retardation in the migration pattern was dependent on SnCl2 concentration and might be due to binding of stannous ions to plasmid DNA. The modification in DNA net electrical charge, due to the presence of bound cationic tin could change DNA migration velocity. In the case of another cation, iron, its binding to DNA was suggested (Meneghini, 1988). In our conditions, both stannous ions bound to DNA and ROS generated by stannous ions close to DNA, could be responsible for alterations in the double strand conformation, which would lead to modifications of the migration pattern. In another set of experiments, plasmid DNA was incubated with stannous chloride in a nitrogen atmosphere for 30 min. The incubation of plasmid DNA with SnCl2 under nitrogen atmosphere diminished the number of DNA SSB, when compared with the injuries observed under
162
J.C.P. de Mattos et al. / Toxicology Letters 116 (2000) 159–163
Fig. 4. Agarose gel electrophoresis of plasmid DNA (pUC 9.1, 200 ng) treated with 200 mg/ml SnCl2 for 30 min in air and nitrogen atmospheres. Lanes: (1) control–air atmosphere; (2) SnCl2 – air atmosphere; (3) SnCl2 –nitrogen atmosphere; (4) control – nitrogen atmosphere; (5) l Hind III DNA, 250 ng.
air atmosphere (Fig. 4, lanes 2 and 3). The quantification of supercoiled form is in agreement with these data (Table 1). A protective effect was also observed when sodium benzoate, which is a hydroxyl radical scavenger, is added to the medium (data not shown), indicating once more the involvement of oxygen radical species in damage induction. This hypothesis is reinforced if we consider that the ionization potentials of Sn(II)/ Sn(III) and Fe(II)/Fe(III) are nearly the same (McLean et al., 1983) and both ions, tin and iron, may be close to DNA. Although 30 min under nitrogen atmosphere would not be sufficient to deplete all oxygen molecules dissolved in water, the different amounts of damaged plasmids obtained in air and nitrogen atmospheres suggest a direct reaction of DNA with stannous chloride. In summary, data presented in this paper indicate that stannous ion has a powerful damaging effect, through oxygen radical formation and suggest its direct binding to DNA. In view of these results we are further encouraged to study other possible effects on deoxyribonucleic acid, which might make SnCl2 a danger to human health.
References Assis, M.L.B., Caceres, M.R., De Mattos, J.C.P., Caldeira-deArau´jo, A., Bernardo-Filho, M., 1998. Cellular inactivation induced by a radiopharmaceutical kit: role of stannous chloride. Toxicol. Lett. 99, 199–205.
Bernardo-Filho, M., Cunha, M.C., Valsa, J.O., Caldeira-deArau´jo, A., Silva, F.C.P., Fonseca, A.S., 1994. Evaluation of potential genotoxicity of stannous chloride: Inactivation, filamentation and lysogenic induction of Escherichia coli. Food Chem. Toxicol. 32, 477 – 479. Budavery, S. (Ed.), 1996. The Merck Index. Merck, Whitehouse Station, NJ, pp. 1500 – 1501. Cabral, R.E.C., Leita˜o, A., Lage, C., Caldeira-de-Arau´jo, A., Bernardo-Filho, M., Dantas, F.J.S., Cabral-Neto, J., 1998. Mutational potentiality of stannous chloride: an important reducing agent in the 99mTc-radiopharmaceuticals. Mutat. Res. 408, 129 – 135. Caldeira-de-Arau´jo, A., Dantas, F.J.S., Moraes, M.O., Felzenszwalb, I., Bernardo-Filho, M., 1996. Stannous chloride participates in the generation of reactive oxygen species. J. Braz. Assoc. Adv. Sci. 48, 109 – 113. Dantas, F.J.S., Moraes, M.O., Carvalho, E.F., Valsa, J.O., Bernardo-Filho, M., Caldeira-de-Arau´jo, A., 1996. Lethality induced by stannous chloride on Escherichia coli AB 1157: Participation of reactive oxygen species. Food Chem. Toxicol. 34, 959 – 962. Dantas, F.J.S., Moraes, M.O., De Mattos, J.C.P., Bezerra, R.J.A.C., Carvalho, E.F., Bernardo-Filho, M., Caldeirade-Arau´jo, A., 1999. Stannous chloride mediates single strand breaks in plasmid DNA through reactive oxygen species formation. Toxicol. Lett. 110, 129 – 136. Devasagayam, T.P.A., Subramanian, M., Singh, B.B., Ramanathan, R., Das, N.P., 1995. Protection of plasmid pBR322 DNA by flavanoids against single-strand breaks induced by singlet molecular oxygen. J. Photochem. Photobiol. B 30, 97 – 103. Felzenszwalb, I., De Mattos, J.C.P., Bernardo-Filho, M., Caldeira-de-Arau´jo, A., 1998. Shark cartilage-containing preparation: protection against reactive oxygen species. Food Chem. Toxicol. 36, 1079 – 1084. Ferguson, L.R., 1994. Antimutagens as cancer chemopreventive agent in the diet. Mutat. Res. 317, 395 – 410. Hallas, L.E., Cooney, J.J., 1981. Tin and tin-resistant microorganisms in Chesapeake Bay. Appl. Environ. Microbiol. 41, 466 – 471. Hattori, T., Maehashi, H., 1994. Augmentation of calcium influx by stannous chloride at mouse motor nerve terminals. Res. Commun. Chem. Pathol. Pharmacol. 84, 253 – 256. Johnson, M.A., Baier, M.J., Greger, J.L., 1982. Effects of dietary tin on zinc, copper, iron, manganese, and magnesium metabolism in adult males. Am. J. Clin. Nutr. 35, 1332 – 1338. Larsson, A., Kinnby, B., Kunsberg, R., Peszkowski, M.J., Warfuinge, G., 1990. Irritant and sensitizing potential of copper, mercury and tin salts in experimental contact stomatitis of rat oral mucosa. Contact Dermatitis 23, 146 – 153. Loeb, L., James, E.A., Natadorph, A.M., Klebanoff, S., 1988. Mutagenesis by autooxidation of iron with isolated DNA. Proc. Natl. Acad. Sci. USA 85, 3918 – 3922.
J.C.P. de Mattos et al. / Toxicology Letters 116 (2000) 159–163 McLean, J.R.N., Blakey, D.H., Douglas, G.R., Kaplan, J.R., 1983. The effect of stannous and stannic (tin) chloride on DNA in Chinese hamster ovary cells. Mutat. Res. 119, 195 – 201. Meneghini, R., 1988. Genotoxicity of active oxygen species in mammalian cells. Mutat. Res. 195, 215–230. Rader, J.I., 1991. Anti-nutritive effects of dietary tin. Adv. Exp. Med. Biol. 289, 509–524. Reiniger, I.W., Silva, C.R.S., Felzenszwalb, I., De Mattos, J.C.P., Oliveira, J.F., Dantas, F.J.S., Bezerra, R.J.A.C., Caldeira-de-Arau´jo, A., Bernardo-Filho, M., 1999. Boldine action against the stannous chloride effect. J. Ethnopharmacol. 68, 345 – 348.
.
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Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Shi, X., Mao, Y., Knapton, A.D., Ding, M., Rojanasakul, Y., Gannet, P., Dalal, N., Liu, K., 1994. Reaction of Cr(VI) with hydrogen peroxide generates hydroxyl radicals and causes DNA damage: role of a Cr(IV)-mediated Fentonlike reaction. Carcinogenesis 15, 2475 – 2478. Simpson, J.A., Cheeseman, K.H., Smith, S.E., Dean, R.T., 1988. Free-radical generation by copper ions and hydrogen peroxide. J. Bacteriol. 254, 519 – 523. White, D.J., 1995. ‘Return’ to stannous fluoride dentifrices. J. Clin. Dent. VI (Spec. Issue), 29 – 36.
Damage induced by stannous chloride in plasmid DNA Jose´ C.P. de Mattos a, Fla´vio J.S. Dantas a, Roberto J.A.C. Bezerra a, Mario Bernardo-Filho a, Janua´rio B. Cabral-Neto b, Claudia Lage b, Alvaro C. Leita˜o b, Adriano Caldeira-de-Arau´jo a,* a
Departamento de Biofı´sica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Uni6ersidade do Estado do Rio de Janeiro, A6. 28 de Setembro, 87, Rio de Janeiro, RJ 20551 -030, Brazil b Laborato´rio de Radiobiologia Molecular, IBCCF, CCS, UFRJ, Rio de Janeiro, RJ 21949 -900, Brazil Received 6 March 2000; received in revised form 2 May 2000; accepted 3 May 2000
Abstract Stannous chloride (SnCl2) is widely used in daily human life, for example, to conserve soft drinks, in food manufacturing and biocidal preparations. In nuclear medicine, stannous chloride is used as a reducing agent of Technetium-99m, a radionuclide used to label different cells and molecules. In spite of this, stannous chloride is able to generate reactive oxygen species (ROS) which can damage DNA. In this work, plasmid DNA (pUC 9.1) was incubated with SnCl2 under different conditions and the results analyzed through DNA migration in agarose gel electrophoresis. Our data reinforce the powerful damaging effect induced by stannous ion and suggest that this salt can play a direct role in inducing DNA lesions. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Stannous chloride; Reactive oxygen species; DNA damage; Plasmid DNA
1. Introduction Reactive oxygen species (ROS) can be generated by several physical and chemical agents. The mono-electronic reduction of ground state oxygen (which occurs during ATP synthesis) causes the appearance of oxygen free radicals, which are implicated in the induction of DNA lesions (Meneghini, 1988; Ferguson, 1994). In view of the importance of DNA damage in carcinogenesis, it is reasonable to suppose that any agent capable of * Corresponding author. Fax: +55-21-2543532. E-mail address: [email protected] (A. Caldeira-de-Arau´jo).
reacting with DNA and modifying its structure could be potentially carcinogenic. Thus, oxidative DNA lesions would account for increasing risks of cancer development. Studies on the genotoxic properties of SnCl2 revealed that it can generate ROS and breaks in DNA (Caldeira-de-Arau´jo et al., 1996) and induces lethality in Escherichia coli, whose damage recovery depends on RecA-mediated repair (Bernardo-Filho et al., 1994). Humans are exposed to stannous chloride (SnCl2) present in packaged food, soft drinks, biocides, dentifrices, etc. (Hallas and Cooney, 1981; McLean et al., 1983; Rader, 1991; White,
0378-4274/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 0 ) 0 0 2 1 3 - 7
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J.C.P. de Mattos et al. / Toxicology Letters 116 (2000) 159–163
1995; Budavery, 1996). In addition, SnCl2 is used in nuclear medicine procedures to obtain Technetium-99m-labelled radiopharmaceuticals (Bernardo-Filho et al., 1994; Caldeira-de-Arau´jo et al., 1996). ROS like hydroxyl radicals (OH) can be produced through the Fenton reaction, i.e. the reduction of superoxide anion (O2−) by Fe2 + ions. Cu2 + and other metal ions are also able to produce O2− and OH through a Fenton-like reaction (Loeb et al., 1988; Simpson et al., 1988; Shi et al., 1994). Dantas et al. (1996) showed that SnCl2-induced lesions could also be generated by a Fenton-like reaction. Moreover, stannous ions have mutagenic effects, which are suggested to occur mainly via DNA oxidation and production of 8-hydroxyguanine (8-oxoG) (Cabral et al., 1998). In addition, Caldeira-de-Arau´jo et al. (1996) showed increased survival of E. coli treated with SnCl2 in the presence of catalase, ROS scavengers or metal ion chelators. In light of these data, we decided to investigate, at a molecular level, using isolated pUC 9.1 plasmid DNA, the possible direct and indirect effects of stannous ions. Conformational changes induced by SnCl2 in plasmid DNA were analyzed in agarose gel electrophoresis. This protocol had already been used to demonstrate the induction of single strand breaks (SSB) by singlet oxygen (Devasagayam et al., 1995). Our data are in accordance with the hypothesis that oxidative lesions can be induced by SnCl2, but also suggest a kind of damage that may be induced by tin itself.
Fig. 1. Agarose gel electrophoresis of plasmid DNA (pUC 9.1, 200 ng) treated with 200 mg/ml SnCl2 for different periods of time. Lanes: (1) control; (2) 30 min; (3) 60 min (4) l Hind III DNA, 250 ng.
2. Material and methods Plasmid DNA (pUC 9.1) was obtained by alkaline lysis as described in Sambrook et al. (1989) and used elsewhere (Caldeira-de-Arau´jo et al., 1996; Assis et al., 1998; Felzenszwalb et al., 1998). Electrophoresis were carried out in 0.8% agarose gels (Sambrook et al., 1989) as previously described (Caldeira-de-Arau´jo et al., 1996; Assis et al., 1998; Felzenszwalb et al., 1998). Plasmid DNA was treated with SnCl2.2H2O (Sigma, St Louis, MO) at 200 mg/ml, for varying periods of time, at room temperature, according to the experimental purpose. Hind III restriction enzyme (Gibco BRL, USA) was used to linearize plasmid DNA when necessary. In some experiments, nitrogen atmosphere was employed to diminish the amount of ROS induced by SnCl2. Ultrapure water (Milli-Q system) was used as solvent. Aliquots of each sample (200 ng DNA) were applied in a horizontal gel plate and electrophoresed at 6 V/cm. The gels were stained with ethidium bromide (0.5 mg/ml) and photographs were taken with a Polaroid camera. Each assay was repeated, at least, three times, the best photos were scanned and the bands quantified through the Gel-Pro Analyzer 3.0 computer program.
3. Results and discussion Several unwanted effects of stannous chloride have been described in the literature (Johnson et al., 1982; Larsson et al., 1990; Hattori and Maehashi, 1994). In the past few years, cytotoxic and genotoxic SnCl2-induced damage were demonstrated in E. coli and the effects appeared to be mediated by reactive oxygen species (Caldeira-deArau´jo et al., 1996; Dantas et al., 1996; Assis et al., 1998; Felzenszwalb et al., 1998; Dantas et al., 1999; Reiniger et al., 1999). Results shown in Fig. 1 indicate that SnCl2 modifies plasmid DNA conformational structure (supercoiled form I changed to open circle form II) in a time-dependent manner. Conversely, if SnCl2 is allowed to undergo pre-oxidation, before it is added to DNA, the number of induced plasmid DNA SSB decreases. This effect was seen
J.C.P. de Mattos et al. / Toxicology Letters 116 (2000) 159–163
Fig. 2. Agarose gel electrophoresis of plasmid DNA (pUC 9.1, 200 ng) treated with 200 mg/ml SnCl2 for 30 min after different periods of exposure of stannous chloride solution to air. Lanes: (1) control; (2) 0 h; (3) 2 h; (4) 4 h; (5) 24 h; (6) l Hind III DNA, 250 ng. Table 1 Quantification of supercoiled form through Gel-Pro Analyzer computer programa Percent of supercoiled form in lane
Fig. 1 Fig. 2 Fig. 4
1
2
3
4
5
100.00 100.00 100.00
49.10 32.10 30.66
41.20 35.20 41.00
– 40.68 100.00
– 75.15 –
a Photos of the gels were scanned and the form I was quantified using a l Hind III DNA, 250 ng as standard. Control samples were normalized as 100% supercoiled form.
Fig. 3. Agarose gel electrophoresis of plasmid DNA (pUC 9.1, 200 ng) linearized with Hind III restriction enzyme and treated with different SnCl2 concentrations for 1 h. Lanes: (1) control; (2) 50 mg/ml SnCl2; (3) 200 mg/ml SnCl2; (4) 400 mg/ml SnCl2; (5) 800 mg/ml SnCl2; (6) 1600 mg/ml SnCl2; (7) control; (8) molecular weight standard DNA (200 ng).
in an agarose gel electrophoresis of SnCl2-treated DNA after exposure of stannous chloride solution to air for different periods of time (Fig. 2). The percentage of supercoiled form I after each experimental situation was quantified by means of a
161
computer program (Gel-Pro Analyzer) and the data are presented in Table 1. These results (Figs. 1 and 2), combined with those from Table 1, can be explained if we assume that stannous chloride has oxidative properties. The ROS generated were responsible for the increase in the number of DNA breaks, in a timedependent manner, as shown in Fig. 1 and confirmed by the decrease in the percentage of supercoiled form (Table 1). The number of DNA SSB decreased when the stannous chloride solution was maintained at room temperature before incubation with plasmid DNA, once more showing the importance of ROS in the induction of DNA lesions (Fig. 2 and Table 1). A stannous chloride pre-oxidation period in periods of time lower than 2 h did not change plasmid DNA electrophoretic profiles (data not shown). An interesting observation that can be seen in Fig. 2 (lane 5) is that even after 24 h of pre-oxidation of the SnCl2 solution, there is a residual amount of DNA lesions in plasmid DNA. It suggests another kind of way that lesions could be produced through a possible direct effect of SnCl2. In order to clarify this point, we carried out experiments in which pUC 9.1 plasmid DNA was first linearized with Hind III restriction enzyme and then treated with increasing concentrations of SnCl2. Fig. 3 shows a crescent delay in DNA migration through agarose gel with increasing amounts of SnCl2. The retardation in the migration pattern was dependent on SnCl2 concentration and might be due to binding of stannous ions to plasmid DNA. The modification in DNA net electrical charge, due to the presence of bound cationic tin could change DNA migration velocity. In the case of another cation, iron, its binding to DNA was suggested (Meneghini, 1988). In our conditions, both stannous ions bound to DNA and ROS generated by stannous ions close to DNA, could be responsible for alterations in the double strand conformation, which would lead to modifications of the migration pattern. In another set of experiments, plasmid DNA was incubated with stannous chloride in a nitrogen atmosphere for 30 min. The incubation of plasmid DNA with SnCl2 under nitrogen atmosphere diminished the number of DNA SSB, when compared with the injuries observed under
162
J.C.P. de Mattos et al. / Toxicology Letters 116 (2000) 159–163
Fig. 4. Agarose gel electrophoresis of plasmid DNA (pUC 9.1, 200 ng) treated with 200 mg/ml SnCl2 for 30 min in air and nitrogen atmospheres. Lanes: (1) control–air atmosphere; (2) SnCl2 – air atmosphere; (3) SnCl2 –nitrogen atmosphere; (4) control – nitrogen atmosphere; (5) l Hind III DNA, 250 ng.
air atmosphere (Fig. 4, lanes 2 and 3). The quantification of supercoiled form is in agreement with these data (Table 1). A protective effect was also observed when sodium benzoate, which is a hydroxyl radical scavenger, is added to the medium (data not shown), indicating once more the involvement of oxygen radical species in damage induction. This hypothesis is reinforced if we consider that the ionization potentials of Sn(II)/ Sn(III) and Fe(II)/Fe(III) are nearly the same (McLean et al., 1983) and both ions, tin and iron, may be close to DNA. Although 30 min under nitrogen atmosphere would not be sufficient to deplete all oxygen molecules dissolved in water, the different amounts of damaged plasmids obtained in air and nitrogen atmospheres suggest a direct reaction of DNA with stannous chloride. In summary, data presented in this paper indicate that stannous ion has a powerful damaging effect, through oxygen radical formation and suggest its direct binding to DNA. In view of these results we are further encouraged to study other possible effects on deoxyribonucleic acid, which might make SnCl2 a danger to human health.
References Assis, M.L.B., Caceres, M.R., De Mattos, J.C.P., Caldeira-deArau´jo, A., Bernardo-Filho, M., 1998. Cellular inactivation induced by a radiopharmaceutical kit: role of stannous chloride. Toxicol. Lett. 99, 199–205.
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